This laboratory has been examining the effects of cysteine substitution and modification in the 5-HT transporter (SERT). We will continue these efforts by examining two regions of the transporter where modeling efforts suggest that sequence previously identified as belonging to transmembrane domains may be in external or internal loops. In the region containing external loop 1 (EL1) and transmembrane domain 2 (TM2), we will test the prediction that TM2 crosses the membrane in a beta configuration and EL1 is consequently larger than predicted. By determining where the patterns of reactivity and activity modification are most consistent with the transition between EL1 and TM2, we hope to distinguish between the original model of SERT topology and the current predictions. We have shown that internal cysteine residues of SERT react with MTSEA-biotin with cells are permeabilized with digitonin, even if non external cysteines are available. We propose to use this property to determine exposure of internal loop residues by substituting the seven-cysteine residues predicted to lie in internal loops. We expect to generate a form of SERT that is unreactive toward MTSEA-biotin both in intact cells and in membrane preparations. Replacing the original cysteine in internal loops (IL) such as IL5 should increase the reactivity of SERT toward MTSEA-biotin. We will test the exposure of residues in the region of TM10 and IL5 to the cytoplasm by replacing residues one at a time with cysteine, and testing for digitonin-dependent reactivity toward MTSEA-biotin and functional consequences of modification. A considerable degree of uncertainty exists in the topological modeling of this region. We hope to establish this method to determine exposure of internal loop domains and to apply to the topology of SERT in the TM10-IL5 region.